Measurement of total, intracellular and surface bound cations in animal cells grown in culture

Abstract

A procedure is described in which animal cells grown in culture on a dish are rapidly rinsed in situ with 0.25 M sucrose solutions for subsequent measurement of total, intracellular and rapidly exchangeab le Na+, K+, Mg2+ and Ca2+ by atomic absorption spectrophotometry. Repeated rinses with CO2-free (pH similar to 7) 0.25 M sucrose solution produced essentially no loss of cellular protein or cations. One 10-second rinse with CO2-saturated (pH 4) 0.25 M sucrose solution removed a rapidly proton exchangeable cellular cation fraction which is interpreted as being externally (membrane) bound. Rinses with physiological electrolyte solutions are shown to produce loss of cellular protein as well as displacement of surface exchangeable cations. Thus, isotonic sucrose solution is more satisfactory than electrolytic media for rinsing cultured cells prior to measurement of cellular cations. The technique employing sucrose rinse media is very rapid and reproducible and permits measurement of total, intracellular or surface bound Na+, K+, Mg2+ and Ca2+ in the same sample.